The complement system is part of the innate immune system and consists of many components that act in a cascade fashion. This system plays a central role in both the clearance of immune complexes and the immune response to infectious agents, foreign antigens, virus-infected cells and tumor cells. However, complement is also involved in pathological inflammation and in autoimmune diseases. Therefore, inhibition of excessive or uncontrolled activation of the complement cascade could provide clinical benefit to patients with such diseases and conditions.
The complement system can be activated in three ways, either by one of the two primary activation pathways, designated the classical and the alternative pathways (V. M. Holers, In Clinical Immunology: Principles and Practice, ed. R. R. Rich, Mosby Press, 1996, 363-391), or by a third pathway, the lectin pathway activated by mannan-binding lectin (MBL) (M. Matsushita, Microbiol. Immunol., 1996, 40: 887-893; M. Matsushita et al., Immunobiol., 1998, 199: 340-347; T. Vorup-Jensen et al., Immunobiol., 1998, 199: 348-357).
The classical pathway is a calcium/magnesium-dependent cascade, which is normally activated by the formation of antigen-antibody complexes. C1, the first enzyme complex in the cascade, is a pentamolecular complex consisting of C1q, 2 C1r molecules, and 2 C1 s molecules. This complex binds to an antigen-antibody complex through the C1q domain to initiate the cascade. Once activated, C1s cleaves C4 resulting in C4b, which in turn binds C2. C2 is cleaved by C1s, resulting in the activated form, C2a, bound to C4b and forming the classical pathway C3 convertase.
The alternative pathway is a magnesium-dependent cascade and is antibody-independent. This pathway is activated by a variety of diverse substances including, e.g., cell wall polysaccharides of yeast and bacteria, and certain biopolymer materials. When the C3 protein binds on certain susceptible surfaces, it is cleaved to yield C3b thus initiating an amplification loop.
The lectin pathway involves complement activation by MBL through two serum serine proteases designated MASP-I and MASP-2 (as opposed to C1r and C1s in the classical complement pathway). Like the classical complement pathway, the lectin complement pathway also requires C4 and C2 for activation of C3 and other terminal components further downstream in the cascade (C. Suankratay et al., J. Immunol., 1998, 160: 3006-3013; Y. Zhang et al., Immunopharmacol., 1999, 42: 81-90; Y. Zhang et al., Immunol., 1999, 97: 686-692; C. Suankratay et al., Clin. Exp. Immunol., 1999, 117: 442-448). Alternative pathway amplification is also required for lectin pathway hemolysis in human serum (C. Suankratay et al., J. Immunol., 1998, 160: 3006-3013; C. Suankratay et al., Clin. Exp. Immunol., 1998, 113: 353-359). In short, Ca++-dependent binding of MBL to a mannan-coated surface triggers activation of C3 following C4 and C2 activation, and the downstream activation of C3 and the terminal complement components then require the alternative complement pathway for amplification.
Activation of the complement pathway generates biologically active fragments of complement proteins, e.g. C3a, C4a and C5a anaphylatoxins and sC5b-9 membrane attack complex (MAC), which mediate inflammatory activities involving leukocyte chemotaxis, activation of macrophages, neutrophils, platelets, mast cells and endothelial cells, vascular permeability, cytolysis, and tissue injury
(R. Schindler et at., Blood, 1990, 76: 1631-1638; T. Wiedmer, Blood, 1991, 78: 2880-2886; M. P. Fletcher et al., Am. J. Physiol., 1993, 265: 111750-1761).
C2 is a single-chain plasma protein of molecular weight of 102 kD, which is specific for the classical and the lectin complement pathways. Membrane bound C4b expresses a binding site which, in the presence of Mg++, binds the proenzyme C2 near its amino terminus and presents it for cleavage by C1s (for the classical complement pathway) or MASP-2 (for the lectin complement pathway) to yield a 30 kD amino-terminal fragment, C2b, and a 70 kD carboxy-terminal fragment, C2a (S. Nagasawa et al., Proc. Natl. Acad. Sci. (USA), 1977, 74: 2998-3003). The C2b fragment may be released or remain loosely attached to C4b. The C2a fragment remains attached to C4b to form the C4b2a complex, the catalytic components of the C3 and C5 convertases of the classical and the lectin complement pathways. The enzymatic activity in this complex resides entirely in C2a, C4b acting to tether C2a to the activating surface.
Monoclonal antibodies (MAbs) to human C2 and its fragments C2a and C2b were made by immunizing mice with purified human C2 (E. I. Stenbaek et al., Mol. Immunol., 1986, 23: 879-886; T. J. Oglesby et al., J. Immunol., 1988, 141: 926-932). The novel anti-C2a MAbs of the present invention were made by immunizing mice with purified human C2a fragment and were shown to have inhibitory activity against the classical pathway complement activation (see below). These anti-C2a MAbs are distinct from the known anti-C2b MAb (see T. J. Oglesby et al., J. Immunol., 1988, 141: 926-932) because they bind to different segments of C2 and inhibit the classical complement pathway by interfering the interaction between C2 and C4 (T. J. Oglesby et al., J. Immunol., 1988, 141: 926-932). By virtue of this inhibition, the anti-C2a MAbs of the present invention are the first Mab demonstrated to be effective in inhibiting the classical complement pathway.
Targeting C2a and/or the C2a portion of C2 for complete inhibition of the classical and the lectin complement pathways has several advantages including, for example: (1) C2 and C2a are specific for the classical and the lectin complement pathways, and thus inhibition of C2 and/or C2a would achieve complete and selective inhibition of these two complement pathways without affecting the alternative complement pathway; (2) the concentration of C2 in human blood is one of the lowest (ca. 20 μg/ml) among other soluble complement components, therefore inhibitors of C2 or C2a would have a unique dose advantage; and (3) since C2a is the catalytic subunit of the C3 and C5 convertases, inhibition of C2 or the C2a portion of C2 would block the activation of C3 and C5.
The down-regulation of complement activation has been demonstrated to be effective in treating several disease indications in animal models and in ex vivo studies, e.g., systemic lupus erythematosus and glomerulonephritis (Y. Wang et al., Proc. Natl. Acad. Sci. (USA), 1996, 93: 8563-8568), rheumatoid arthritis (Y. Wang et al., Proc. Natl. Acad. Sci. (USA), 1995, 92: 8955-8959), in preventing inflammation associated with cardiopulmonary bypass and hemodialysis (C. S. Rinder et al., J. Clin. Invest., 1995, 96: 1564-1572; J. C. K. Fitch et al., Circulation, 1999, 100: 2499-2506; H. L. Lazar et al., Circulation, 1999, 100: 1438-1442), hyperacute rejection in organ transplantation (T. J. Kroshus et al., Transplantation, 1995, 60: 1194-1202), myocardial infarction (J. W. Homeister et al., J. Immunol., 1993, 150: 1055-1064; H. F. Weisman et al., Science, 1990, 249: 146-151), reperfusion injury (E. A. Amsterdam et al., Am. J. Physiol., 1995, 268: H448-H457), and adult respiratory distress syndrome (R. Rabinovici et al., J. Immunol., 1992, 149: 1744-1750). In addition, other inflammatory conditions and autoimmune/immune complex diseases are also closely associated with complement activation (V. M. Holers, ibid., B. P. Morgan. Eur. J. Clin. Invest., 1994, 24: 219-228), including thermal injury, severe asthma, anaphylactic shock, bowel inflammation, urticaria, angioedema, vasculitis, multiple sclerosis, psoriasis, dermatomyositis, myasthenia gravis, membranoproliferative glomerulonephritis, and Sjögren's syndrome.